An Improved Method For Constructing Mouse Tongue Cancer Animal Model And RNA Sequencing Study Of Tongue Cancer

Objective:To establish an animal model of tongue squamous cell carcinoma in mice fed with 4-nitroquinoline-1-oxide water,mimicking the behavior of human chewing betel nut,and to observe whether an animal model of tongue squamous cell carcinoma with high cancer inducing rate and mimicking the development of human disease was obtained.The differentially expressed genes were annotated and analyzed by transcriptome sequencing technology to explore the potential therapeutic targets of tongue cancer.Methods:(1)Sixty female C57BL/6J mice were selected to establish tongue cancer animal models.The mice were randomly divided into a water-fed group(N = 6)and a control group(n = 6).Four groups were fed with 4NQO water(n = 6 each).There were 6 rats in each group in experimental group G1(4NQO water feeding combined with arecoline mechanical application).Experimental group G2(arecoline mechanical application)6 rats.Groups C and G1 were randomly sacrificed at the end of 12,16,20 and 24 weeks,respectively.The pathological changes were observed by gross examination and histopathology,and statistical analysis was performed.The changes of the lingual mucosa in the normal group N and the simple arecoline mechanical application group G2 were observed.The animals were sacrificed immediately if abnormal proliferation was found,and the final death time was 24 weeks if there was no abnormal proliferation.(2)At the end of 20 weeks,4NQO water feeding combined with arecoline mechanical application group G1 successfully established an animal model of tongue cancer,which was significantly different from that of the control group C.RNA-seq technology was used to sequence the gene expression of C and G1 groups compared with the normal group N group at the end of 20 weeks.The common differential genes between the two groups were selected as the target differential gene set,and the correlation between these differential genes and pathways in the occurrence of tongue cancer was analyzed by bioinformatics methods.Because C and G1 groups have different cancer-inducing pathways,RNA-seq technology was performed to analyze the cancer-promoting pathways and key gene expression of arecoline in these two groups.Results:(1)At 20 weeks,only 1 of 6 mice in group C developed cancer,and the cancer-inducing rate was 17%.All the 6 mice in G1 group were cancerous,and the carcinogenic rate was 100%.In G2 group,only arecoline mechanical application group,no abnormal hyperplasia was observed in the lingual mucosa during the whole experiment,and the rats were killed at the end of the experiment at the end of the 24 th week.Only one case of mild abnormal hyperplasia was found in the pathological examination.(2)The C and G1 groups were selected for sequencing at the end of 20 weeks,respectively,and there were 310 differentially expressed genes in the two groups,which were used as the target gene set.The target gene set was enriched in the metabolic pathways related to the progression of tongue cancer and the C-type lectin receptor signaling pathway.GO and KEGG enrichment analysis showed that arecoline promoted carcinogenesis was related to UDP-N-acetylglucosamine metabolism and mucin-type O-glycan pathway.Conclusion:(1)The 4NQO combined with arecoline mechanical coating group successfully established the animal model of tongue cancer at the end of 20 weeks,which was4 weeks earlier than that in the conventional 4NQO water feeding group,and the incidence of tongue cancer reached 100%.This improved method successfully established an efficient tongue cancer animal model.(2)During the progression of tongue cancer,it is related to the immune genes of IL17 F and IL17 A in IL-17 signaling pathway.Meanwhile,C-type lectin receptor signaling pathway was found to be highly enriched during the progression of tongue cancer,indicating that the progression of tongue cancer may be closely related to tumor immune response,which provides the basis for further research on the mechanism of tongue cancer.(3)This study found that arecoline is related to glycosylation-related metabolic pathways in the process of promoting the occurrence of cancer,and controls the metabolic process of udp-n-acetylglucose by down-regulating GFPT1 gene,thus inhibiting the apoptosis of cancer cells.They play an important role in the proliferation,apoptosis,invasion and metastasis of tumors,which may be the reason why arecoline promotes the occurrence of cancer.